Studies On The Cloning And Analysis Of Galactose-1-phosphate Uridylyltransferase Gene Of Gracilariopsis Lemaneiformis And The Correlation Between Gene Expression And Agar Synthesis

Gracilaria /Gracilariopsis lemaneiformis is a red macroalga which belongs to Rhodophyta, Gigartinales, Gracilariaceae.Attributed to the controversial argument about the classification of Gracilaria lemaneiformis, there is still a doubt about whether it belongs to Gracilariopsis or Gracilaria. Gurgel(1991) identifies three monophyletic clades in the Gracilariaceae including Melanthalia andCurdiea, Gracil ario psis, Gracilaria. In this studies 5.8S rDNA– ITS region of individuals within the population of Gracilaria lemaneiformis were amplified and analyzed. The whole sequence was 1066bp in length, including 250 bp of ITS1, 159bp of 5.8S and 657bp of ITS2. There was no genetic difference within the population of Gracilaria lemaneiformis. The rDNA-ITS sequences of 20 Gracilaria and Gracilariopsis species indexed from NCBI data base was compared and phylogenic analyzed. The result indicated that the sequences of 5.8S were highly conserved among Gracilaria lemaneiformis and 19 Gracilariaceae species. However, the ITS regions were various in sequence and its length range. The genetic distances between 20 Gracilariaceae species were between 0.013 and 0.596. Gracilaria lemaneiformis was much similar to Gracilariopsis species than to Gracilaria species referring to the 5.8S rDNA– ITS characters. The UPGMA phylogenic tree was constructed of the 20 Gracilariaceae species. The result indicated that Gracilaria lemaneiformis of Qingdao strain should be included in the genus Gracilariopsis, which evolved earlier from the family Gracilariaceae. Gracilaria lemaneiformis was situated at an ancient position within the clade.The most economical significance of Gracilaria is the agar. It has attracted much more attention for its high agar content and quality. As a major economy species of Gracilariopsis, it is important to research on the agar correlation gene using molecular techniques, to understand the agar metabolism mechanism and regulations. In this studies, galactose-1-phosphate uridylyltransferase, a key enzyme in the process of agar synthesis was selected as the target. The primer was designed according to the galt sequence from close species. RNA was extracted, the cDNA was polyadenylated and amplified by PCR, the part of galt sequence was obtained. The primer was obtained according to the part sequence, reverse transcription and PCR amplification of the 3′end was performed using RACE. After handling splicing, the full length cDNA of galt 1240 bp long including untranslated 151 bp 3'- flanking region and an open reading frame of 362 amino acids. The full length DNA of galt 1447 bp long was deduced from PCR and invitro cloning results. RpsBLAST analysis indicated that both of the C and N ends of the protein had conservative combine location with Zn2+ and Fe2+ , and also there was a HIT element which was essential to enzyme activity. There was also a highly conserve sequence comprised the active site between 166 and 175 amino acid. The sequence information was analyzed in details and the result of Southern blotting indicated that galt has only one copy in Gracilaria lemaneiformis genome.Investigated agar content from Gracilaria lemaneiformis of the same nature population, found that distances were shown in the production of agar among different individuals. Choosing two groups within the biggest discrepancy, after RNA extracting and cDNA reversing, the gene expression efficiency with different agar content was evaluated by real-time PCR. data denoted for the comparative expression. indicating that there is relativity between agar content and galt gene expression, the lower agar content with higher galt gene expression, higher agar conten with higher expression in most case. However because of lower GALT protein ,lower enzyme activity or effect of other enzyme in the pathway of agar synthesis ,there is no direct ratio between the galt gene expression and agar content .The GALT may not be the key enzyme in the agar synthsis path way The content of agar is not controlled by GALT directly. But, the result also indicated that the GALT gene expression and the agar content with highly relativity. Because the research is absent in agar synthesis, so screen out the key enzyme needs father investigation.